Association between HLA-C alleles and COVID-19 severity in a pilot study with a Spanish Mediterranean Caucasian cohort

The clinical presentations of COVID-19 may range from an asymptomatic or mild infection to a critical or fatal disease. Several host factors such as elderly age, male gender, and previous comorbidities seem to be involved in the most severe outcomes, but also an impaired immune response that causes a hyperinflammatory state but is unable to clear the infection. In order to get further understanding about this impaired immune response, we aimed to determine the association of specific HLA alleles with different clinical presentations of COVID-19. Therefore, we analyzed HLA Class I and II, as well as KIR gene sequences, in 72 individuals with Spanish Mediterranean Caucasian ethnicity who presented mild, severe, or critical COVID-19, according to their clinical characteristics and management. This cohort was recruited in Madrid (Spain) during the first and second pandemic waves between April and October 2020. There were no significant differences in HLA-A or HLA-B alleles among groups. However, despite the small sample size, we found that HLA-C alleles from group C1 HLA-C*08:02, -C*12:03, or -C*16:01 were more frequently associated in individuals with mild COVID-19 (43.8%) than in individuals with severe (8.3%; p = 0.0030; pc = 0.033) and critical (16.1%; p = 0.0014; pc = 0.0154) disease. C1 alleles are supposed to be highly efficient to present peptides to T cells, and HLA-C*12:03 may present a high number of verified epitopes from abundant SARS-CoV-2 proteins M, N, and S, thereby being allegedly able to trigger an efficient antiviral response. On the contrary, C2 alleles are usually poorly expressed on the cell surface due to low association with β2-microglobulin (β2M) and peptides, which may impede the adequate formation of stable HLA-C/β2M/peptide heterotrimers. Consequently, this pilot study described significant differences in the presence of specific HLA-C1 alleles in individuals with different clinical presentations of COVID-19, thereby suggesting that HLA haplotyping could be valuable to get further understanding in the underlying mechanisms of the impaired immune response during critical COVID-19.

Q2. Please describe the clinical protocols that the authors used for the defining of mild, severe and critical COVID-19 patients, did the authors follow the WHO guideline for example SpO2 level, etcs?
A2. We agree with Reviewer#1 that this is an essential question. Patients were classified depending on the medical assistance they received and according to the progression of the disease. Mild COVID-19 received primary health assistance from Primary Healthcare Centers, whereas severe and critical COVID-19 were hospitalized. Critical COVID-19 were admitted to the Intensive Care Unit (ICU). This information was included in lines 279-289. This classification was also based on the WHO's recommendations (World Health Organization. (2021). Living guidance for clinical management of COVID-19 [Ebook]. Retrieved from https://www.who.int/publications/i/item/WHO/2019-nCoV/clinical/2021.2). We also include Tables S1 and S2 to clarify the data.
Q3. Please perform a multiple correction adjustment for all the p-values in the HLA association analysis, for example the HLA-B*14:01 would not be significant after multiple correction; P=0.023*12, thus there are no HLA alleles significantly associate with the severity of COVID-19 and the title and the writings of the paper should reflect as such.
A3. We thank Reviewer#1 for this critical observation. We agree with the necessity of performing this analysis and therefore, P values have been corrected using Bonferroni correction after alleles with an expected value <5 in each group had been grouped as "rare alleles". For each HLA haplotype the corrected P value (Pc) was calculated by multiplying the P value obtained with the two-tailed Chi-square or Fisher's exact test by the number of tested allelic combinations. Only Pc values lower than 0.05 were considered to be statistically significant. Significant differences regarding HLA-C*08:02 are maintained between Mild and Critical COVID-19 (pc=0.0240) (Table  2). Similarly, HLA-C*08:02 or HLA-C*12:03 or HLA-C*16:01 are significantly more frequent in Mild group than in Severe (pc=0.033) and Critical (pc=0.0154) groups (Table 3). In addition, HLA-C*06:02 or HLA-C*07:01 are significantly more prevalent in the Severe group regarding Mild COVID-19 (pc=0.0132) ( Table 3). After Bonferroni correction, no statistical significance was observed in HLA haplotypes (Table 4) or in allele HLA-B*14:01. We have modified the manuscript accordingly, adding several sentences highlighted in red throughout the text.
Q4. The obvious disadvantage of this paper are the tiny sample size used in this study, the lack of healthy control, the lack of replication sample set, the lack of appropriate adjustment for multiple testing.
A4. We understand the concern raised by Reviewer#1 about this limitation of our study. We previously discuss this issue in lines 645-652. We would like to indicate, first, that we attained statistical significance in some comparison, despite the reduced sample size. And second, that other studies that evaluate the association of HLA with COVID-19 severity is a small sample have been published previously (Iturrieta-Zuazo I, et al. 2020;Lorente L, et al. 2021;Wang W, et al 2020;Abdelhafiz AS, et al.2021). Besides, these cohorts of individuals with COVID-19 prevaccination era are very precious as this situation is unlikely to be repeated again, due to more than 95% of the Spanish population and 65% of world population have at least received one dose of the authorized vaccines. Finally, we also indicated in the manuscript that this observational study is consider a pilot study, and the results would need to be confirmed in a larger cohort.
The aim of this study was to find a possible association between HLA alleles and COVID-19 severity. Because of that, a healthy group was not considered, similarly to previous reports with a similar objective (Iturrieta-Zuazo I, et al. 2020). Moreover, the frequency of the different HLA alleles in the Spanish population have been mentioned in the introduction (Montero-Martín G, et al.2019) (Lines 123-124) and discussed (Montero-Martín G, et al.2019;Arnaiz-Villena A, et al. 1997. Martinez-Laso J, et al. 1995529-530;538-539;546-552;553-556).
We also agree with Reviewer#1 that the lack of multiple adjustment was necessary to obtain significant results. According to Reviewer#1 suggestion, we applied Bonferroni correction to adjust p values, as explained above.
Q5. The authors performed extra HLA association test by HLA carrier frequencies, but most of the significant alleles are either very rare (example: less than 5), these results should be gathered and put in a combined group such as the rare HLA group.
A5. We agree with Reviewer#1 that this suggestion is also necessary to clarify data expressed in Table 2 and also contributes to get a better understanding of the results. Consequently, those alleles with an expected value <5 in each group have been grouped as "rare alleles" for every HLA class I and class II genes tested. Next, Bonferroni correction was performed, considering this new group of "rare alleles" as one determination.

Minor comments
Q1. "Most of these alleles concentrate the highest rate of mutations in the exon that encodes the peptide-binding pocket, likely due to the selection pressure exerted by infectious diseases during pandemics". The reason why mutations have been reported in the exon are mainly caused by the fact that conventional HLA genotyping method such as PCR-SSOP often only targets exon2.3 of the HLA genes, causing a bias in the reporting of exonic variants in the IMGT/HLA database, in fact, the recent database is now full of intronic/3'UTR/5'UTR variants. A1. We thank Reviewer#1 for this suggestion. In order to avoid misunderstandings, we believe that is better to delete this sentence.